Indian green mussels (Perna viridis) are the cheap source of proteins and considered as a delicacy. Extract prepared from green mussels by enzyme-acid hydrolysis process showed various biological activities. In our earlier patent (US patent application #20030044470) we have shown the inhibition of osteoclast differentiation and activation in the crude extract. In same continuation, attempts have been made to purify the active compound that showed inhibition of osteoclast differentiation and activation. The purification of the crude extract was done using HPLC, gel filtration and TLC methods. The purified compound was again checked for the above activity. The compound was characterized using NMR and LC-MS/MS techniques. This compound was synthesized and checked for the presence of the above biological activity. This patent in particular describes the synthesis of the compound and also its activity for inhibition of osteoclast formation.
Novel class of amino acid/dicarboxylic acid derivatives (sulfonic acid/sulfate derivatives of naturally occurring amino acids and their amides) along with calcium is for their activation to show inhibition of the osteoclastogenesis. Amino acid derivatives and calcium ion when used separately did not show any activity on inhibition of the osteoclastogenesis. The following classes of compounds are identified    (1) Natural acidic amino acids (Aspartic acid, Glutamic acid and their amides),    (2) Unnatural amino acids, amides such as homoglutamic acid,    (3) Dicarboxylic acids such as succinic acid, glutaric acid, and adipic acid    (4) N-sulfonyl, C-sulfonyl/sulfate derivatives of the above acids    (5) Alkaline earth metals such as Mg, Zn and Ca as their suitable salts.Related Arts
A lot of information is available on the matrix metalloproteinases (MMP's) commonly used as MMP inhibitors for the treatment of osteoporosis (Nigel, R. A. Beeley, Phillip, R. J., Ansell, Andrew, J. P., Dochert, 1994, Curr. Opin. Ther. Patents., 4, 7-16). A cylinder shaped solid compound has been prepared from the atelocollagen solution, L-alanine solution and bone morphogenetic protein for treating bone loss and elevating bone deformities (Hiroo, Akhihiko, Rebecca, Wozney, Seeherman, 2003, WO Patent #2003066083). In another study glutamate and glutamate derivatives/analogs or their mixtures have been used for modulating the bone quality (Tadeusz, Jose Luis; Stefan, 2003, WO Patent # 2003043626). Toshhiro (2003) invented a compound consisting of interacting trans-activators with glutamic acid, aspartic acid and rich carboxyl-terminal domain for estrogen receptor dependent activity (Toshihiro, 2002, WO Patent # 2003000730). Glutamic acid has been defined as an effective neuromediator and one of its derivatives is involved in osteoclast formation and bone resorption. The modification of glutamic acid action in bone could be used for bone remodeling (Hopital E. Herriot, Lyon Fr., 2002, Microscopy Research and technique, 58(2), 70-76).
However, these inhibitors have various problems and efforts were made for the development of non-peptide inhibitors. For instance in the specification of EP 606046, several aryl-sulfonamide derivatives have been described. In another invention several aryl sulfonyl amino acid derivatives of the following specifications have been described (Sakaki, Kanazawa, Sugiura, Miyazaki, Ohno, 2002, U.S. Pat. No. 6,403,644) as MMP inhibitors:    1) N-[[4-(Benzoylamino)phenyl]sulfonyl]glycine,    2) N-[[3-(Benzoylamino)phenyl]sulfonyl]glycine,    3) N-[[2-(Benzoylamino)phenyl]sulfonyl]glycine,    4) N-[[4-(Acetylamino)phenyl]sulfonyl]glycine,    5) N-[[4-(Phenylacetylamino)phenyl]sulfonyl]glycine,    6) N-[[4-[(Phenylethylcarbonyl)amino]phenyl]sulfonyl]glycine,    7) N-[[4-(Cinnamoylamino)phenyl]sulfonyl]glycine,    8) N-[[4-(N-Phenylureido)phenyl]sulfonyl]glycine,    9) N-[[4-(N-Phenylthioureido)phenyl]sulfonyl]glycine,    10) N-[[4-[(Benzyloxycarbonyl)amino]phenyl]sulfonyl]glycine,    11) N-[[4-[(Phenyloxymethylcarbonyl)amino]phenyl]sulfonyl]glycine,    12) N-[[4-[(Benzyloxymethylcarbonyl)amino]phenyl]sulfonyl]glycine,    13) N-[[4-(4-Methoxybenzoylamino)phenyl]sulfonyl]glycine,    14) N-[[4-(4-Fluorobenzoylamino)phenyl]sulfonyl]glycine,    15) N-[[4-(4-Nitrobenzoylamino)phenyl]sulfonyl]glycine,    16) N-[[4-(3-Nitrobenzoylamino)phenyl]sulfonyl]glycine,    17) N-[[4-(2-Nitrobenzoylamino)phenyl]sulfonyl]glycine,    18) N -[[4-(4-Formylbenzoylamino)phenyl]sulfonyl]glycine,    19) N-[[4-(Benzoylamino)phenyl]sulfonyl]-D-alpha-phenylglycine,    20) N-[[4-(Benzoylamino)phenyl]sulfonyl]-L-alpha-phenylglycine,    21) N-[[4-(4-Methylbenzoylamino)phenyl]sulfonyl]-D-alpha-phenylglycine,    22) N-[[4-(Methylbenzoylamino)phenyl]sulfonyl]-L-alpha-phenylglycine,    23) N-[[4-(4-Methoxybenzoylamino)phenyl]sulfonyl]-D-alpha-phenylglycine,    24) N-[[4-(4-Methoxybenzoylamino)phenyl]sulfonyl]-L-alpha-phenylglycine,    25) N-[[4-(4-Fluorobenzoylamino)phenyl]sulfonyl]-D-alpha-phenylglycine,    26) N-[[4-(4-Fluorobenzoylamino)phenyl]sulfonyl]-L-alpha-phenylglycine,    27) N-[[4-(4-Nitrobenzoylamino)phenyl]sulfonyl]-D-alpha-phenylglycine,    28) N-[[4-(4-Nitrobenzoylamino)phenyl]sulfonyl]-L-alpha-phenylglycine,    29) N -[(4-Pivaloyloxyphenyl)sulfonyl]-D,L-alpha-phenylglycine,    30) N-[(4-Pivaloyloxyphenyl)sulfonyl]-D,L-phenylalanine,    31) N-[[4-(2,4-Dichlorobenzoylamino)phenyl]sulfonyl]glycine,    32) N-[[4-(2,4-Dichlorobenzoylamino)phenyl]sulfonyl]-D,L-alanine,    33) N-[[4-(2,4-Dichlorobenzoylamino)phenyl]sulfonyl]-beta-alanine,    34) N-[[4-(2,4-Dichlorobenzoylamino)phenyl]sulfonyl]-L-valine,    35) N-[[4-(2,4-Dichlorobenzoylamino)phenyl]sulfonyl]-D,L-valine,    36) N-[[4-(2,4-Dichlorobenzoylamino)phenyl]sulfonyl]-L-Ieucine,    37) N-[[4-(2,4-Dichlorobenzoylamino)phenyl]sulfonyl]-D,L-Ieucine,    38) N-[[4-(2,4-Dichlorobenzoylamino)phenyl]sulfonyl]-D,L-serine,    39) N-[[4-(2,4-Dichlorobenzoylamino)phenyl]sulfonyl]-L-phenylalanine,    40) N-[[4-(2,4-Dichlorobenzoylamino)phenyl]sulfonyl]-L-tyrosine,    41) N-[[4-(2,4-Dichlorobenzoylamino)phenyl]sulfonyl]-D,L-alanine methyl ester,    42) N-[[4-(2,4-Dichlorobenzoylamino)phenyl]sulfonyl]-L-valine methyl ester,    43) N-[[4-(2,4-Dichlorobenzoylamino)phenyl]sulfonyl]-D,L-valine methyl ester,    44) N-[[4-(2,4-Dichlorobenzoylamino)phenyl]sulfonyl]-L-Ieucine methyl ester,    45) N-[[4-(2,4-Dichlorobenzoylamino)phenyl]sulfonyl]-D,L-serine methyl ester,    46) N-[[4-(2,4-Dichlorobenzoylamino)phenyl]sulfonyl]-L-tyrosine methyl ester,    47) N-[[4-(3-Nitrobenzoylamino)phenyl]sulfonyl]-L-aspartic acid,    48) N-[[3-(3-Nitrobenzoylamino)phenyl]sulfonyl]-L-aspartic acid,    49) N-[[4-(3-Aminobenzoylamino)phenyl]sulfonyl]-L-aspartic acid,    50) N-[[3-(3-Aminobenzoylamino)phenyl]sulfonyl]-L-aspartic acid,    51) N-[[4-(Benzoylamino)phenyl]sulfonyl]-L-glutamic acid,    52) N-[[4-(4-Chlorobenzoylamino)phenyl]sulfonyl]-L-glutamic acid,    53) N-[[4-(4-Nitrobenzoylamino)phenyl]sulfonyl]-L-glutamic acid,    54) N-[[4-[2-(4-(1-Pyrrolidinyl)phenyl]butyryloxy]phenyl]sulfonyl]-D,L-3-morpholinoalanine ethyl ester,    55) N-[[4-[2-(4-(1-Nitrophenyl)butyryloxy]phenyl]sulfonyl]-D,L-3-morpholinoalanine ethyl ester,    56) N-[[4-(2-Methoxy-2-phenylacetyloxy)phenyl]sulfonyl]-D,L-3-morpholinoalanine ethyl ester,    57) N-[[4-[[[1-(4-Nitrophenyl)cyclobutyl]carbonyl]oxy]phenyl]sulfonyl]-D,L-3-morpholinoalanine ethyl ester,    58) N-[[3-Methyl-4-[2-[4-(1-Pyrrolidinyl)phenyl]butyryloxy]phenyl]sulfonyl]-t-butoxycar-L-lysine,    59) N-[[4-(2-Phenylbutyryloxy)phenyl]sulfonyl]glycine,    60) N-[[4-[2-[4-(1-Pyrrolidinyl)phenyl]butyryloxy]phenyl]sulfonyl]-D,L-phenylalanine,    61) N-[[4-[2-[4-(1-Pyrrolidinyl)phenyl]butyryloxy]phenyl]sulfonyl]-D,L-aspartic acid,    62) N-[[4-[[[1-(4-Nitrophenyl)cyclobutyl]carbonyl]oxy]phenyl]sulfonyl]-D,L-aspartic acid,    63)1-[[4-[2-[4-(1-Pyrrolidinyl)phenyl]butyryloxy]phenyl]sulfonylamide]-1-cyclopropanecarboxylic acid,    64) N-[[4-[2-[4-(1-Pyrrolidinyl)phenyl]butyryloxy]phenyl]sulfonyl]-D,L-2-(2-furanyl)glycine,    65) N-[[4-[2-[4-(1-Pyrrolidinyl)phenyl]butyryloxy]phenyl]sulfonyl]-D,L-2-(2-tri-enyl)glycine,    66) N-[[4-[2-[4-(1-Pyrrolidinyl)phenyl]butyryloxy]phenyl]sulfonyl]-L-valine,    67) N-[[4-[2-[4-(1-Pyrrolidinyl)phenyl]butyryloxy]phenyl]sulfonyl]-S-carboxymethyl-L-cysteine,    68) N-[[4-[2-Ethyl-2-(4-methoxyphenyl)butyryloxy]phenyl]sulfonyl]-glycine,    69) N-[[3-Methyl-4-[2-[4-(1-Pyrrolidinyl)phenyl]butyryloxy]phenyl]sulfonyl]-L-lysine,    70) N-[[3 Methyl-4-[2-[4-(1-pyrrolidinyl)phenyl]butylyloxy]phenyl]sulfonyl]amino]pentanoic acid,    71) N-[[(3-Methyl-4-pivaloyloxy)phenyl]sulfonyl]-beta-alanine.Purpose of the Invention
Bone is a metabolically active and highly organized connective tissue. The main functions of the bones are provision of mechanical and structural support, maintaining blood calcium levels, supporting haematopoiesis and housing the important vital organs such as brain, spinal cord and heart. To accomplish these functions bone needs continuous remodeling. Bone contains two distinct cell types, the osteoblasts, essential for bone formation (synthesis); and the osteoclasts, essential for bone resorption (break down). Morphogenesis and remodeling of bone involves the synthesis of bone matrix by osteoblasts and coordinated resorption by osteoclasts. The co-ordination between the osteoblasts and osteoclasts is very crucial in maintaining bone homeostasis and structural integrity of the skeleton. Both these processes are influenced by several hormones and local factors generated within bone and bone marrow, resulting in a complex network of control mechanisms. An imbalance of osteoblast and osteoclast functions can result in skeletal abnormalities characterized by increased or decreased bone mass. This may leads to excessive bone loss that reflects the balance of bone formation and bone resorption. Bone destruction or resorption is carried out by haematopoietically derived osteoclasts. Their number and activity is determined by cell lineage allocation, proliferation and differentiation of osteoclast precursors and the resorptive efficiency of mature osteoclasts. Important bone diseases such as osteoporosis, rheumatoid arthritis, Paget's disease of bone and bone metastasis of breast and prostate cancers are caused by increased osteoclast activity (Teitelbaum, 2000). In these disorders bone resorption exceeds bone formation resulting in decreased skeletal mass. This causes bones to become thin, fragile and susceptible to fracture. The consequences of osteoporotic bone fractures include chronic pain in bone, body deformity including height loss and muscle weakness. Therefore, to understand both pathogenesis and successful treatment of these bone diseases there is a need for better understanding of biology of osteoclasts.
Osteoporosis is now a serious problem that imposes substantial limitations on the affected individuals. In human, 1 in 3 women and 1 in 12 men over 45 years are at risk of suffering painful and deforming fractures as a result of osteoporosis. More women die after hip fractures than from cancers of ovaries, cervix and uterus. Osteoporosis occurs at a relatively earlier age in Indian males and females compared to western countries (Gupta, 1996). A variety of disadvantages are associated with current therapeutic agents used in osteoporosis and other metabolic bone disorders. The side effects of current therapies include increase in the risk of breast and uterine cancers, upper gastrointestinal distress and induction of immune responses. Drugs that inhibit the formation or activity of osteoclasts and with no toxicity and harmful side effects will be valuable for treating osteoporosis and other bone diseases. Bone resorption and loss of calcium from bone are complications associated with arthritis, many cancers and with bone metastases of breast and prostate tumors. Because of lack of research into osteoporosis and related diseases, we don't know all the answers to treat these diseases. Progress in better understanding the pathogenesis and successful treatment of these diseases to date has targeted osteoclast. Osteoclasts, the only cells capable of resorbing bone differentiate from the haemopoietic precursors of monocyte/macrophage lineage that also give rise to macrophages and dendritic cells (Miyamoto et al. 2001). Lineage choice and the differentiation process is guided by lineage restricted key regulatory molecules and transcription factors. Osteoclasts are large multinucleated cells. They are formed by the fusion of mononuclear cells of haemopoietic origin and not by mitosis, since DNA synthesis is not required. Osteoclast formation and bone resorption is regulated by many hormones, growth factors and immune cell-derived cytokines (Udagawa et al. 1995, Wani et al. 1999, Fox et al. 2000, Fuller et al. 2000). These factors act directly or indirectly via other cell types to influence osteoclast differentiation. The most important cell type influencing osteoclast formation is osteoblast, which promote this process by a contact-dependent mechanism. Recent advances revealed that two molecules, macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) expressed by osteoblasts are essential and sufficient for the differentiation of haemopoietic cells to form osteoclasts (Tanaka et al. 1993, Anderson et al., 1997, Wong et al. 1997, Lacey et al. 1998, Yasuda et al. 1998). The precise role of other cells, such as T lymphocytes in bone homeostasis is yet to be fully elucidated. It has recently been reported that activated T cells regulates osteoclast formation by some unknown mechanisms. T cells support osteoclast formation by RANKL-dependent and RANKL-independent mechanisms (Weitzmann et al. 2001). Cytokines produced by activated T cells, as well as by other cell types regulates osteoclastogenesis in physiological and pathological conditions. Recent discovery of RANKL has enabled us for the meticulous dissection of mechanisms by which various factors regulate osteoclastogenesis, and better understanding of both pathogenesis and successful treatment bone diseases. In our preliminary studies, we have investigated the role of novel compounds on osteoclastogenesis induced by RANKL in the presence of M-CSF in stromal cell-free cultures of osteoclast precursors.
Natural products from plants and organisms have frequently been used as a source for development of effective drugs. There is an increased interest in analysis of natural products from marine organisms. Sea animals contain metabolites which can be used for treatment of many diseases.
The inventors have previously shown (US Patent #2003066083) that a novel extract (mussel hydrolysate) prepared from the Indian green mussel (Perna viridis) inhibits the osteoclast differentiation in murine haemopoietic precursors of monocyte/macrophage cell lineage. The extract also inhibits the bone resorbing activity of osteoclasts. There was approximately 80-90% inhibition of osteoclast formation and bone resorption in the presence of extract. More importantly, the extract is non-toxic to other cells and is useful to prepare a drug for the treatment of osteoporosis and other bone diseases.
In further investigation, at each stages of purification we found a significant inhibition of osteoclast formation and bone resorption (60-90%). We have purified some active components from extract and these active components significantly inhibit both osteoclast formation and bone resorption. These active compounds can be used in therapeutic settings to protect and cure the individuals against osteoporosis and other metabolic bone diseases.
The current known therapeutic agents have a variety of associated disadvantages. The side effects of current therapies include an elevated risk of breast and uterine cancers, upper gastrointestinal distress and induction of immune responses (Watts' 1999). Our earlier US Patent (#2003066083) describes preparation of mussell hydrolysate from the Indian green mussel (Perna viridis) and its inhibition of the osteoclast differentiation in murine hemopoietic precursors of monocyte/macrophage cell lineage. The extract also shows inhibition of the bone resorbing activity of osteoclasts.
The present inventors have found that a series of novel sulfonic acid/sulfate derivatives of acidic amino acids, aspartic acids, glutamic acid, homoglutamic acid and their related aliphatic dicarboxylic acids (Succinic acid, glutaric acid and adipic acid) have inhibitory activity against osteoclast formation and bone resorption. These compounds are novel and non-toxic to other cells. The active compounds may play a vital role in inhibition of differentiation of osteoclast from hemopoietic precursors and can be used in therapeutic settings to protect and cure the individuals against osteoporosis and other metabolic bone diseases.